Conducting airway epithelia are the primary target of inhaled air pollutants that include tobacco smoke. The cellular responses to the inhalation-related injury are quite different among different regions of airways. In trachea and large airway regions, the expression of squamous epithelium (squamous cell metaplasia) is a predominant feature in these lining cells. The mechanisms involved in this change are still unknown. In this proposal, we focus on the regulation of the expression of a squamous cell differentiation marker, small proline-rich protein (spr1), by cigarette smoke in airway epithelial cells. We hypothesize that spr1 gene expression is an early marker of squamous cell differentiation and the regulation of this gene expression by cigarette smoke occurs at the transcriptional level. Specifically, we will compare the effects of environmental tobacco smoke (ETS) and mainstream tobacco smoke (MTS) on the expression of the spr1 gene in primary cultures of monkey/human tracheobronchial epithelial cells that are maintained in a biphasic culture system (Specific Aim 1). We will determine the nature of this regulation at both the transcriptional and post-transcriptional levels (Specific Aim 2). These studies will lead to the characterization of both the cis-elements and trans-factors involved in the regulation (Specific Aim 3). Once these tobacco smoke-responsive elements are identified, we will determine the effects of various known chemicals that are present in tobacco smoke, such as nitrosamine, nicotine, CO, NO, and several carcinogens (Specific Aim 4). These studies will lead to a better understanding of the nature of gene regulation by tobacco smoke. Preliminary studies demonstrated that an increase of spr1 gene expression is closely associated with airway cell squamous differentiation. We recently isolated both the full-length cDNA and the genomic clones of the human spr1 gene. An antibody, which is monospecific to the spr1 protein, has been developed. Using these probes, we demonstrated that vitamin A down-regulates spr1 mRNA level through decreasing the stability of mRNA in cultured cells, while a phorbol ester, phorbol 12-myristate 12-acetate (PMA), up-regulates spr1 gene expression at the transcriptional level. This up-regulation by PMA is associated with the AP-1 binding site found in the 5'-regulatory region of the spr1 gene. A similar approach should be able to determine the nature of cigarette smoke on the regulation of the expression of the squamous cell marker spr1.